Detection of DNA sequences such as DNA sequences within genes has been used for various purposes including research and disease diagnosis. Compounds useful for detecting DNA sequences include DNA and RNA oligonucleotide probes, typically labeled with some detectable marker such as a radioisotope, a fluorescent dye, or an immunogenic peptide. Nevertheless, there is a continuing need for compounds which can be employed for such purposes. The compounds of the present invention fulfill this need.
Certain of the compounds of this invention may also be used to regulate transcription and expression of genes including genes the regulated expression of which is implicated in, or can be useful in, the treatment or amelioration of disease.
Correct regulation of gene expression is required for fundamental processes in differentiation and development. [See Davidson, E. H., Gene Activity in Early Development, Edn. Third. (Academic Press, Inc., Orlando; 1986)]. In a number of cases, the transcriptional onset and decline of a series of closely related genes are tightly and sequentially controlled, a process that is critical for attaining the correct genotypic readout and proper phenotypic effect. [See Bresnick, E. H., Martowicz, M. L., Pal, S. & Johnson, K. D., Developmental control via GATA factor interplay at chromatin domains. J Cell Physiol 205, 1-9 (2005), Stamatoyannopoulos, G., The molecular basis of blood diseases, Edn. 3rd. (W.B. Saunders, Philadelphia; 2001); and Krumlauf, R, Noordermeer D., de Laat W., Joining the loops: β-globin gene regulation. IUMMB Life 60, 824-833 (2008); and Krumlauf, R., Hox genes in vertebrate development. Cell 78, 191-201 (1994).]A particularly well-studied example of this process is the developmental control of the hemoglobin proteins, particularly those encoded by the genes within the β-like globin locus. [See Stamatoyannopoulos, G., The molecular basis of blood diseases, Edn. 3rd. (W.B. Saunders, Philadelphia; 2001); Krumlauf, R., Hox genes in vertebrate development. Cell 78, 191-201 (1994); and Schechter, A. N., Hemoglobin research and the origins of molecular medicine. Blood 112, 3927-3938 (2008).] These variants exhibit a sequential erythroid-restricted pattern of expression during development, beginning with the yolk sac ε-globin, switching to the fetal γ-globin, and ending with the adult β-globin.
The critical requirement for correct regulation of this locus is demonstrated by the moderate to life-threatening clinical manifestations exhibited by the β-thalassemias. β-thalassemia is primarily caused by mutations in the β-globin gene that lead to reduced or complete loss of β-globin expression. Along with other hemoglobinopathies (such as sickle cell disease), they give rise to the most common single gene genetic disorder worldwide. [See Weatherall, D. J., in The Molecular Bases of Blood Diseases. (eds. G. Stamatoyannopoulos, A. W. Nienhuis, P. W. Majerus & H. Varmus) 207-205 (W.B. Saunders Co., Philadelphia; 1994).] Pharmacological reactivation of the silent fetal (γ) globin chain provides a therapeutic benefit to these patients by compensating for absent adult β-globin chains (in β-thalassemia) or by interfering with the polymerization of mutant hemoglobins (in sickle cell disease); however, these are not always free from complications. [See Weatherall D. J., in The Molecular Bases of Blood Diseases. (eds. G. Stamatoyannopoulos, A. W. Nienhuis, P. W. Majerus & H. Varmus) 157-256 (W. B. Saunders Co., Philadelphia; 1994); and Atweh, G. F. & Schechter, A. N., Pharmacologic induction of fetal hemoglobin: raising the therapeutic bar in sickle cell disease. Curr Opin Hematol 8, 123-130 (2001).] As a result, there remain compelling reasons to search for novel approaches and reagents that achieve reactivation with low toxicity and high penetrance.
Regulation of gene expression is also of particular importance for the generation of induced pluripotent stem (iPS) cells. iPS cells are pluripotent stem cells expressing many of the genetic and phenotypic characteristics of embryonic stem (ES) cells. iPS cells have the same gross morphology as ES cells, proliferative properties, form teratomas after transplantation into nude mice, and have the ability to differentiate along all 3 germ layers in vitro. Their responses to key factors such as retinoic acid and leukemia inhibitory factor (LIF) are also the same as those observed for ES cells [Okita et al, Generation of germline-competent induced pluripotent stem cells. Nature 448: 313-317 (2007)]. Generation of iPS cells is useful for both in vitro study of stem cells (e.g., factors controlling stem cell differentiation) and for the application of iPS cells for the treatment of disease. For example, the ability to reprogram cells from human blood would be useful for the generation of patient-specific stem cells for treatment of diseases in which the disease-causing somatic mutations are restricted to cells of the hematopoietic lineage.
Currently, methods for generating iPS cells from somatic cells are limited, and novel methods are needed. iPS cells have the potential to revolutionize medicine, as they can theoretically generate any differentiated tissue from the self-same individual and thus avoid tissue rejection and other complications. An original protocol for generating iPS cells established iPS cells from murine and human fibroblasts by introducing four specific transcription factors, SOX2, OCT4, KLF4, and c-MYC, into the cells by viral transduction. [See, Lowry, W. E., et al. Generation of human induced pluripotent stem cells from dermal fibroblasts. Proc. Natl. Acad. Sci. USA 105, 2883-2888 (2008); Maherali, N., et al. Directly reprogrammed fibroblasts show global epigenetic remodeling and widespread tissue contribution. Cell Stem Cell; 1, 55-70 (2007); Park, et al. Reprogramming of human somatic cells to pluripotency with defined factors. Nature 451, 141-146 (2008); Takahashi, K., and Yamanaka, S. Induction of Pluripotent Stem cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors. Cell, 126, 663-676 (2006); Takahashi, K. et al. Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors. Cell 131, 861-872 (2007); Yu, J. et al. Induced pluripotent stem cell lines derived from human somatic cells. Science; 318, 1917-1920 (2007); Stadtfeld and Hochedlinger, Induced pluripotency: history, mechanisms, and applications. Genes Dev; 24:2239-2263 (2010); and Hochedlinger and Plath, Epigenetic reprogramming and induced pluripotency. 136; 509-523 (2009).]
Recently, iPS cells were generated from CD34+ mobilized human peripheral blood cells using retroviral transduction of OCT4, SOX2, KLF4, and c-MYC [see, Loh, Y. et al. Generation of induced pluripotent stem cells from human blood. Blood; 113(22):5476-5479 (2009)], and from CD133+ human cord blood (CB) cells [see, Giorgetti, A. et al. Generation of induced pluripotent stem cells from human cord blood using OCT4 and SOX2. Cell Stem Cell; 5:353-357 (2009)]. iPS cells were generated from CD133+ CB cells by retroviral transduction of CB cells with OCT4 and SOX2. CB cells are considered an alternative to bone marrow (BM) as a source of hematopoietic stem cells for transplantation. CB cells can be collected without any risk for the donor, are young cells expected to carry minimal somatic mutations, and possess the immunological immaturity of newborn cells [see, Rocha et al., et al. Transplants of Umbilical-Cord Blood or Bone Marrow from Unrelated Donors in Adults with Acute Leukemia. N. Engl. J. Med. 351, 2276-2285 (2004)]. These properties allow for less stringent criteria for HLA-donor-recipient selection, which represents a decisive benefit for transplantation and has resulted in more than 400,000 immunologically characterized CB units being currently available worldwide through a network of CB banks [see, Gluckman, E., and Rocha, V. Cord blood transplantation: state of the art. Haematologica; 94, 451-454 (2009)].
These approaches brought closer the possibility of using patient-specific cells in cell-based therapy. At least three limitations of those methods, however, were immediately recognizable: (i) the genes were transduced virally; (ii) at least one of the factors, c-Myc, is a known oncogene; and (iii) the process was inefficient. In particular, in the above described studies, retroviral transduction was used to introduce the genes required for iPS cell generation. The use of viruses to deliver the reprogramming factors entails permanent genetic alterations that render the cells inappropriate for many in vitro and in vivo applications. Retroviral gene transduction is associated with a number of limitations (low efficiency), and potential danger for use in human therapy, including the risk of production of replication competent virus, which can infect humans [reviewed in Kurian, K. M. et al. (2000) J Clin Pathol: Mol Pathol; 53:173-176; Stadtfeld and Hochedlinger (2010) Genes Dev; 24:2239-2263].
Thus, what is needed in the art are novel, efficient ways for inducing a selected target gene or genes in a cell without the use of retroviral transduction and without genetically perturbing the recipient cell.
Peptide nucleic acids (PNAs) are oligonucleotide analogues in which the phosphodiester backbone of DNA is replaced by an achiral uncharged polyamide backbone. [See Nielsen, P. E., Egholm, M., Berg, R. H. & Buchardt, O., Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide. Science 254, 1497-1500 (1991).] They are true DNA mimics, as they form Watson-Crick bonds with DNA and RNA, but are of higher thermal stability than natural duplexes due to the lack of electrostatic repulsion. [See Kaihatsu, K., Janowski, B. A. & Corey, D. R., Recognition of chromosomal DNA by PNAs. Chem. Biol 11, 749-758 (2004).] They are also resistant to proteases and nucleases, and thus afford a significantly greater biological stability in culture and in vivo. [See Pooga, M., Land, T., Bartfai, T. & Langel, U., PNA oligomers as tools for specific modulation of gene expression. Biomol Eng 17, 183-192 (2001).]A unique aspect of PNAs is that amino acids can be covalently added to the peptide backbone at either end of the sequence of bases. The PNA/DNA interaction may occur through triple helical (Hoogsteen) base paring (PNA/DNA/PNA) or via a single strand invasion. [See Kaihatsu, K., Janowski, B. A. & Corey, D. R., Recognition of chromosomal DNA by PNAs. Chem. Biol 11, 749-758 (2004); and Zhang, X., Ishihara, T. & Corey, D. R., Strand invasion by mixed base PNAs and a PNA-peptide chimera. Nucleic Acids Res 28, 3332-3338 (2000).] In a single strand invasion, PNA, which can be of mixed sequence design, hybridizes with one strand of DNA through Watson-Crick base pairing and simply replaces the other strand of the double helix.
PNA molecules are thus promising candidates for clinical use as agents to modulate gene expression. For the most part, PNAs have been used as an antigene agent because they have the capacity for down-regulating gene expression in cultured cells and in animals. [See Nielsen, P. E., Peptide nucleic acids as antibacterial agents via the antisense principle. Expert Opin Investig Drugs 10, 331-341 (2001); Cutrona, G. et al., Effects in live cells of a c-myc anti-gene PNA linked to a nuclear localization signal. Nat Biotechnol 18, 300-303 (2000); Janowski, B. A. et al. Inhibiting transcription of chromosomal DNA with antigene peptide nucleic acids. Nat Chem Biol 1, 210-215 (2005); Hu, J. et al., Allele-specific silencing of mutant huntingtin and ataxin-3 genes by targeting expanded CAG repeats in mRNAs. Nat Biotechnol 27, 478-484 (2009); and Nielsen, P. E., PNA Technology. Mol Biotechnol 26, 233-248 (2004).]
PNA molecules have also been used as probes for targeted nucleic acid binding, and to follow the subcellular trafficking of plasmid DNA. Additionally, conjugation of markers such as fluorophores to PNA molecules has been described for these purposes. [See also Zhilina et al., Peptide Nucleic Acid Conjugates: Synthesis, Properties and Applications. Current Topics in Medicinal Chemistry 4:1119-1131 (2005)]. PNA molecules have also been utilized for single base pair mutation analysis by PNA directed PCR clamping. [See also Orum et al., Single base pair mutation analysis by PNA directed PCR clamping. Nucleic Acids Research 21(23):5332-5336 (1993)] However, there is a need for improved PNA structures capable of efficiently detecting the presence of DNA within the nucleus of cells.
PNAs have been modified to maximize cellular/nuclear entry in order to increase the efficiency for in vivo applications. [See Cutrona, G. et al., Effects in live cells of a c-myc anti-gene PNA linked to a nuclear localization signal. Nat Biotechnol 18, 300-303 (2000); Nielsen, P. E., Addressing the challenges of cellular delivery and bioavailability of peptide nucleic acids (PNA). Q Rev Biophys 38, 345-350 (2005); and Braun, K. et al., A biological transporter for the delivery of peptide nucleic acids (PNAs) to the nuclear compartment of living cells. J Mol Biol 318, 237-243 (2002).] However, currently there is no established PNA conjugated system that combines these varied modifications and has been demonstrated to stably and efficiently enter, target, and transcriptionally activate an endogenous chromosomal locus in living cells.
Thus, there remains a great need in the art for novel, efficient and non-toxic compounds which can affect gene expression. Such compounds would be particularly useful in treatment of β-globin disorders, which can be treated by upregulating γ-globin transcription in bone marrow cells, and for the generation of iPS cells, for example by the induction of genes such as OCT4, SOX2, c-MYC, and/or KLF4.